Process for the enzymatic regeneration of redox cofactors

ABSTRACT

A process for the enzymatic regeneration of the redox cofactors NAD + /NADH and NADP + /NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, characterized in that a) in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of general formula R 1 C(O)COOH is reduced, and b) in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, a compound of general formula R 2 CH(OH)R 3  is oxidized and wherein R 1 , R 2  and R 3  in the compounds have different meanings.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of U.S. patent application Ser. No.15/490,416, filed Apr. 18, 2017, which is a division of U.S. patentapplication Ser. No. 14/376,512, filed Aug. 4, 2014, issued U.S. Pat.No. 9,644,227, which is a 371 application of PCT/EP2013/052313, filedFeb. 6, 2013, which claims the benefit of European Patent ApplicationNo. 2450007.5, filed Feb. 7, 2012, International Patent Application No.PCT/IB2012/067781, filed Sep. 12, 2012, and Austrian Patent ApplicationNo. 1284/2012, filed Dec. 10, 2012. The disclosures of the foregoing areincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a process for the enzymaticregeneration of the redox cofactors NAD⁺/NADH and NADP⁺/NADPH in aone-pot reaction, wherein, as a result of at least two furtherenzymatically catalyzed redox reactions proceeding in the same reactionbatch (=product-forming reactions), one of the two redox cofactorsaccumulates in its reduced form and, respectively, the other one in itsoxidized form.

PRIOR ART

Enzymatically catalyzed redox reactions are used in industrialoperations, for example, in the production of chiral alcohols, α-aminoacids and α-hydroxy acids. The majority of enzymes employed inindustrial redox reactions use cofactors such as NADH or NADPH. Amongenzymatic redox reactions, those are particularly interesting whereinredox cofactors are restored by in situ cofactor regeneration systems.The reason therefor is that it is possible to use only catalytic amountsof the expensive cofactors (NAD(P)⁺/NAD(P)H). The availability ofsuitable dehydrogenases and other enzymes has resulted in thedevelopment of various cofactor regeneration systems.

The regeneration systems described up to now may be classified as:enzyme-linked, substrate-linked, in vivo (natural cofactor regenerationsystems in living organisms), photochemical, chemical orelectro-enzymatic. The process herein described relates to anenzyme-linked regeneration system. Advantages of enzyme-linked systemsare high selectivity, applicability for the production of variousproducts and a high reuse rate of the cofactor (total turnover number,TTN).

In the mid-90ies, a first industrial process using an enzyme-linkedcofactor regeneration system was employed on a ton scale. In saidprocess, formate dehydrogenase from Candida boidinii was used. Theindustrial processes yet known normally use a redox enzyme for thesynthesis of the product and a further enzyme for the cofactorregeneration.

Processes wherein two or more enzymatic redox reactions which areinvolved in the formation of the product and two enzymatic systems forthe cofactor regeneration (simultaneously or sequentially) areproceeding in one reaction batch without an intermediate being isolatedmust be distinguished therefrom. Recently, such enzymatic cascadereactions—herein referred to as one-pot reactions—have drawn significantattention, since they effectively reduce operating costs, operating timeand environmental impacts. In addition, enzymatic cascades of redoxreactions facilitate transformations which are not easy to implement byconventional chemical methods.

It is, however, a challenge to perform several reactions (oxidation andreduction) simultaneously in one one-pot reaction with a parallelcofactor regeneration, since highly divergent reaction conditions areoften required for the individual transformations. So far, only a verysmall number of one-pot trials comprising oxidation and reductionreactions with associated cofactor regeneration systems have beenperformed.

In the literature (Advanced Synth. Catal., 2008, Volume 351, Issue 9, p.1303-1311), the experiment of a one-pot reaction using 7α-hydroxysteroiddehydrogenase (HSDH), 7β-HSDH and 12α-HSDH has been described. In saidprocess, an oxidation, both regioselective and stereoselective, wasperformed at positions 7 and 12 of cholic acid, followed by a regio- andstereoselective reduction at position 7. In that process both, a lactatedehydrogenase (NAD⁺-dependent) and a glucose dehydrogenase(NADP⁺-dependent) were used as a cofactor regeneration system. Pyruvateand glucose were used as cosubtrates. Although this process wasoriginally aimed at a true one-pot process, at the end oxidation andreduction reactions were performed separately. In doing so, thepartitioning of oxidative and reductive steps occurred either in aso-called “tea bag”-reactor or in a membrane reactor. Said partitioningwas necessary in order to avoid the production of byproducts due to thelow cofactor selectivity of NADPH-glucose dehydrogenase. However, in theone-pot reaction, the glucose dehydrogenase NADP⁺ converted partly alsoNAD⁺, which impeded the oxidation. In the process described, only 12.5mM (˜0.5%) of the substrate cholic acid was used, which renders theprocess uninteresting from an ecological point of view.

Furthermore, an attempt to perform the deracemization of racemates ofsecondary alcohols via a prochiral ketone as an intermediate using aone-pot system has been described (J. Am. Chem. Soc., 2008, Volume 130,p. 13969-13972). The deracemization of secondary alcohols was achievedvia two alcohol dehydrogenases (S- and R-specific) with differentcofactor specificities. In said system, NADP was regenerated by NADPHoxidase (hydrogen peroxide producing) and NADH was regenerated byformate dehydrogenase. Formate and oxygen were used as cosubstrates. Inthat system 4 enzymes were used without partitioning of oxidative andreductive steps. A drawback of the process is the very low concentrationof the substrate used of 0.2-0.5%, which is inappropriate for industrialpurposes.

A further one-pot system has been described in WO 2009/121785 A2. Insaid process, a stereoisomer of an optically active secondary alcoholwas oxidized to the ketone and then reduced to the corresponding opticalantipode, wherein two alcohol dehydrogenases having oppositestereoselectivities and different cofactor specificities were used. Thecofactors were regenerated by means of a so-called “hydride-transfersystem”, using only one additional enzyme. For regenerating thecofactors, various enzymes such as formate dehydrogenase, glucosedehydrogenase, lactate dehydrogenase were used. A drawback of saidprocess is the low concentration of the substrates used.

A drawback of the enzymatic one-pot methods involving cofactorregeneration systems yet known is altogether the very low substrateconcentration, which is inefficient for industrial processes.

In contrast to that, many individual enzymatic redox reactions arealready known in which cofactor regeneration systems are used. Theexperiments were described with whole microorganisms, cell lysates orisolated enzymes with concurrent NAD(P)H or NAD(P)⁺ regeneration. Knownenzymatic cofactor regeneration systems for individual redox reactionscomprise, for example, formate dehydrogenase for NADH (formate as acosubstrate), alcohol dehydrogenase from Pseudomonas sp. for NADH(2-propanol as a cosubstrate), hydrogenase for NADH and NADPH (H₂ as acosubstrate), glucose-6-phosphate dehydrogenase from L. mesenteroidesfor NADPH (glucose-6-phosphate as a cosubstrate), glucose dehydrogenasefor NADH and NADPH (glucose as a cosubstrate), NADH oxidase for NADH (O₂as a cosubstrate) and phosphite dehydrogenase for NADH (phosphite as acosubstrate).

An example of use of such individual redox reactions is the productionof chiral hydroxy compounds, starting from appropriate prochiral ketocompounds. In said process, the cofactor is regenerated by means of anadditional enzyme. These methods have in common that they constitute anisolated reduction reaction and regenerate NAD(P)H (see e.g. EP 1 152054).

Enzymatic processes using hydroxysteroid dehydrogenases, coupled with acofactor regeneration system, which proceed at higher substrateconcentrations (approx. >1%), have been described (EP 1 731 618; WO2007/118644; Appl. Microbiol. Biotechnol., 2011 Volume 90 p. 127-135).In said processes, the cofactors NAD(P)H or NAD(P) were regenerated bymeans of different enzymes such as, e.g., lactate dehydrogenase(pyruvate as a cosubstrate), alcohol dehydrogenase from T. brockii(isopropanol als as a cosubstrate), alcohol dehydrogenase from L.brevis, L. minor, Leuconostoc carnosum, T. ethanolicus, Clostridiumbeijerinckii. However, these known processes relate merely to theisolated single reactions for the oxidation of a hydroxy compound or forthe reduction of an oxo compound.

A cofactor regeneration system for NADH using malate dehydrogenase(“malate enzyme”) has already been described (Can. J. Chem. Eng. 1992,Volume 70, p. 306-312). In said publication, it was used for thereductive amination of pyruvate by alanine dehydrogenase. The pyruvateemerging during the cofactor regeneration was subsequently used in theproduct-forming reaction.

In WO 2004/022764, it is likewise described to regenerate NADH by malatedehydrogenase. Differently to the previously described publication thepyruvate emerging during the oxidative decarboxylation of malate was notused further.

An example of an enzymatic reduction of D-xylose to xylitol involving acofactor regeneration system has been described (FEBS J., 2005, Volume272, p. 3816-3827). An NADPH-dependent mutant of phosphite dehydrogenasefrom Pseudomonas sp. was used as the cofactor regeneration enzyme. Thisis also a single reaction for the formation of a product.

Further examples of an enzymatic production of chiral enantiomericallyenriched organic compounds, e.g., alcohols or amino acids, have beendescribed (Organic Letters, 2003, Volume 5, p. 3649-3650; U.S. Pat. No.7,163,815; Biochem. Eng. J., 2008, Volume 39(2) p. 319-327; EP 1 285962). In said systems, an NAD(P)H-dependent oxidase from Lactobacillusbrevis or Lactobacillus sanfranciscensis was used as the cofactorregeneration enzyme. The trials likewise constitute single reactions forthe formation of a product.

In WO 2011/000693, a 17beta-hydroxysteroid dehydrogenase as well as aprocess are described enabling the execution of redox reactions atposition 17 of 4-androstene-3,17-dione. Again, this is an isolatedreduction reaction. The above-mentioned individually proceedingoxidation or reduction reactions lack the advantages of a one-potreaction, such as for example cost-effectiveness as a result of time andmaterial savings as well as a better turnover due to enzymatic cascadereactions.

SUMMARY

The object of the present invention was to provide a process forregenerating the redox cofactors NAD⁺/NADH and/or, e.g. and, NADP⁺/NADPHin order to perform therewith two or more enzymatically catalyzed redoxreactions in one reaction batch in an economical fashion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reaction scheme of the epimerization ofchenodeoxycholic acid into ursodeoxycholic acid via the intermediate3α-hydroxy-7oxo-5β-cholanic acid, with cofactor regeneration using2-propanol and pyruvate.

FIG. 2 shows the reaction scheme of the epimerization ofchenodeoxycholic acid into ursodeoxycholic acid via the intermediate3α-hydroxy-7oxo-5β-cholanic acid, with cofactor regeneration usingmalate and pyruvate.

FIG. 3 shows the reaction scheme of the epimerization ofchenodeoxycholic acid into ursodeoxycholic acid via the intermediate3α-hydroxy-7oxo-5β-cholanic acid, with cofactor regeneration using2-propanol and oxygen.

FIG. 4 shows the reaction scheme of the isomerization of glucose intofructose, with cofactor regeneration using 2-propanol and pyruvate.

FIG. 5 shows the reaction scheme of the isomerization of glucose intofructose, with cofactor regeneration using 2-propanol and oxygen.

FIG. 6 shows the reaction scheme of the epimerization of cholanic acidto 3α,7β-dihydroxy-12-oxo-5β-cholanic acid via the intermediates3α,7α-dihydroxy-12-oxo-5β-cholanic acid and3α-hydroxy-7,12-dioxo-5β-cholanic acid with cofactor-regeneration using2-propanol and pyruvate.

FIG. 7 shows the reaction scheme of the epimerization of cholanic acidto 3α,7β-dihydroxy-12-oxo-5β-cholanic acid via the intermediates3α,7α-dihydroxy-12-oxo-5β-cholanic acid and3α-hydroxy-7,12-dioxo-5β-cholanic acid with cofactor-regeneration of theepimerization of cholanic acid to 3α,7β-dihydroxy-12-oxo-5β-cholanicacid via the intermediates 3α,7α-dihydroxy-12-oxo-5β-cholanic acid and3α-hydroxy-7,12-dioxo-5β-cholanic acid with cofactor-regeneration using2-propanol and oxygen.

FIG. 8 and FIG. 9 show the reaction schemes of the epimerization ofcholanic acid to 3α,7β-dihydroxy-12-oxo-5β-cholanic acid via theintermediates 3α,7α-dihydroxy-12-oxo-5β-cholanic acid and3α-hydroxy-7,12-dioxo-5β-cholanic acid with cofactor-regeneration using2-propanol, pyruvate and oxygen.

FIG. 10 shows possible reaction schemes of the epimerization of cholanicacid to 3α,7β-dihydroxy-12-oxo-5β-cholanic acid via differentintermediates and cofactor-regeneration systems. For regeneration ofNAD+ alternatively lactate dehydrogenase (pyruvate as a substrate) andNADH oxidase (oxygen as a substrate) were used. For regeneration ofNADPH alcohol dehydrogenase (isopropanol as a substrate) was used.

FIG. 11 shows the reaction scheme of the epimerization ofchenodeoxycholanic acid to ursodeoxycholanic acid via the intermediate3α-hydroxy-7oxo-5β-cholanic acid (7-ketolithocholanic acid=7K-LCA=KLC)with cofactor regeneration using 2-propanol and 2-pentanol (in each casealcohol dehydrogenase) as well as pyruvate (lactate dehydrogenase) andoxygen (NADH oxidase).

In the figures the following abbreviations are used:

BsSDH=sorbitol dehydrogenase from Bacillus subtilis

CA=3α,7α,12α-trihydroxy-5β-cholanic acid

7β-CA=3α,7β,12α, -trihydroxy-5β-cholanic acid

Caoxo=Clostridium aminovalericum NADH oxidase

CDC, CDCA=3α,7α-dihydroxy-5β-cholanic acid

CtXR=Candida tropicalis xylose reductase

7α-HSDH=7α-hydroxysteroid dehydrogenase

7β-HSDH=7β-hydroxysteroid dehydrogenase

12α-HSDH=12α-hydroxysteroiddehydrogenase

KLC=3α-hydroxy-7-oxo-5β-cholanic acid

7K-LCA=3α-hydroxy-7-oxo-5β-cholanic acid

LacDH=lacate dehydrogenase NAD(H)-dependent

LkADH=Lactobacillus kefir alcohol dehydrogenase NADP(H)-dependent

Lmoxid=Leuconostoc mesenteroides NADH-oxidase

MalDH=E. coli malate dehydrogenase NADP(H)-dependent

7oxo-CA=3α, 12 α-dihydroxy-7-oxo-5β-cholanic acid

12oxo-CDC=3α,7α-dihydroxy-12-oxo-5β-cholanic acid

12oxo-KLC=3α-hydroxy-7,12-dioxo-5β-cholanic acid

12oxo-UDC=3α,7β-dihydroxy-12-oxo-5β-cholanic acid

SISDH=sheep liver sorbitol dehydrogenase

SmOxo=Streptococcus mutans NADH oxidase

UDC. UDCA=3α,7β-dihydroxy-5β-cholanic acid

DETAILED DESCRIPTION

According to the present invention, said object is achieved in a processof the kind initially mentioned, in that a process for the enzymaticregeneration of the redox cofactors NAD⁺/NADH and/or, e.g. and,NADP⁺/NADPH in a one-pot reaction is provided, wherein, as a result ofat least two further enzymatically catalyzed redox reactions proceedingin the same reaction batch (product-forming reactions), one of the tworedox cofactors accumulates in its reduced form and, respectively, theother one in its oxidized form,

which process is characterized in that

-   -   a) in the regeneration reaction which reconverts the reduced        cofactor into its original oxidized form, oxygen or a compound        of general formula

wherein R₁ represents a linear-chain or branched (C₁-C₄)-alkyl group ora (C₁-C₄)-carboxy alkyl group, is reduced, and

-   -   b) in the regeneration reaction which reconverts the oxidized        cofactor into its original reduced form, a (C₄-C₈)-cycloalcanol        or a compound of general formula

wherein R₂ and R₃ are independently selected from the group consistingof H, (C₁-C₆) alkyl, wherein alkyl is linear-chain or branched, (C₁-C₆)alkenyl, wherein alkenyl is linear-chain or branched and comprises oneto three double bonds, aryl, in particular C₆-C₁₂ aryl, carboxyl, or(C₁-C₄) carboxy alkyl, in particular also cycloalkyl, e.g. C₃-C₈cycloalkyl,

is oxidized.

A process provided according to the present invention is herein alsoreferred to as “process according to (of) the present invention”.

In a further aspect, the present invention provides a process accordingto the present invention for the enzymatic regeneration of the redoxcofactors NAD⁺/NADH and/or, e.g. and, NADP⁺/NADPH in a one-pot reaction,wherein, as a result of at least two further enzymatically catalyzedredox reactions proceeding in the same reaction batch (=product-formingreactions), one of the two redox cofactors accumulates in its reducedform and, respectively, the other one in its oxidized form,

which process is characterized in that

-   -   a) during the regeneration of the oxidized cofactor, a compound        of general formula

wherein R₁ represents a substituted or unsubstituted C₁-C₄ alkyl group,is reduced, and

-   -   b) during the regeneration of the reduced cofactor, a compound        of general formula

wherein R₂ and R₃ independently of each other are selected from thegroup consisting of

-   -   1) —H,    -   2) —(C₁-C₆) alkyl, wherein alkyl is linear-chain or branched,    -   3) —(C₁-C₆) alkenyl, wherein alkenyl is linear-chain or branched        and optionally comprises up to three double bonds,    -   4) -cycloalkyl, in particular C₃-C₈ cycloalkyl,    -   5) -aryl, in particular C₆-C₁₂ aryl,    -   6) —(C₁-C₄) carboxy alkyl, in case compound I is pyruvate,        optionally also carboxyl;

is oxidized.

In a further aspect, in a process according to the present invention, R₂and R₃ independently of each other are selected from the groupconsisting of H, (C₁-C₆) alkyl, wherein alkyl is linear-chain orbranched, (C₁-C₆) alkenyl, wherein alkenyl is linear-chain or branchedand comprises one to three double bonds, aryl, in particular C₆-C₁₂aryl, carboxyl, or (C₁-C₄) carboxy alkyl.

Compared to the prior art, a process according to the present inventionconstitutes a significant improvement of processes in which compoundsare both enzymatically oxidized and reduced, since it is enabled to runthe required oxidation and reduction reactions as well as the associatedreactions for the cofactor regeneration in one reaction batch and, atthe same time, to use significantly higher substrate concentrations thanaccording to prior art.

In a process according to the present invention, the cofactors NADHand/or NADPH are used. Therein, NAD⁺ denotes the oxidized form and NADHdenotes the reduced form of nicotinamide adenine dinucleotide, whereasNADP⁺ denotes the oxidized form and NADPH denotes the reduced form ofnicotinamide adenine dinucleotide phosphate.

Enzymatically catalyzed redox reactions which are not part of thecofactor regeneration and, in a process according to the presentinvention, are involved in the formation of the product are hereinreferred to as “oxidation reaction(s)” and “reduction reaction(s)”.“Oxidation reaction(s)” and “reduction reaction(s)” are summarized underthe term “product-forming reactions”. The product-forming reactions in aprocess according to the present invention comprise, in each case, atleast one oxidation reaction and at least one reduction reaction.

If NAD⁺ is used as a cofactor for the oxidation reaction(s), NADPH isthe cofactor for the reduction reaction(s). If NADP⁺ is used as acofactor for the oxidation reaction(s), NADH is the cofactor for thereduction reaction(s).

In a process according to the present invention, oxidation reaction(s)and reduction reaction(s) can be performed either chronologicallyparallel or in chronological succession, preferably chronologicallyparallel in the same reaction batch.

Compounds which are used with the objective of forming a product areherein referred to as substrates. Compounds which are reacted during thecofactor regeneration are herein referred to as cosubstrates.

In a process according to the present invention, one substrate as wellas several substrates can be used. In doing so, reduction and/oroxidation reaction(s) can take place on the same substrate (molecularbackbone) and also on different substrates, preferably on the samesubstrate. Furthermore, in a process according to the present invention,reduction and/or oxidation reactions can take place on the same or ondifferent functional groups.

A process according to the present invention is suitable for a pluralityof reactions, for example for the inversion of configuration ofstereoisomeric hydroxy compounds via oxidation to the correspondingketone and subsequent reduction to the opposite stereospecific hydroxycompound.

A process in which two or more enzymatic redox reactions involved in theformation of a product and two enzymatic systems for cofactorregeneration proceed in one reaction batch without an intermediate beingisolated is herein referred to as a “one-pot reaction”.

The mentioning of an acid or a salt of an acid includes herein therespective unmentioned term. Likewise, the mentioning of acids, inparticular of bile acids, includes herein all esters derived therefrom.Furthermore, compounds (partly) provided with protective groups areincluded in the mentioning of the underlying substances.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that the oxidation reactionand the reduction reaction proceed chronologically parallel.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that both the oxidationreaction and the reduction reaction occur on the same molecularbackbone.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that, as a compound offormula I (2-oxo acid), pyruvate (cosubstrate) is used which is reducedto lactate by means of a lactate dehydrogenase, which means that, in theregeneration reaction which reconverts the reduced cofactor into itsoriginal oxidized form, pyruvate is reduced to lactate by means of alactate dehydrogenase.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that, as a compound offormula II (secondary alcohol), 2-propanol (isopropyl alcohol, IPA)(cosubstrate) is used which is oxidized to acetone by means of analcohol dehydrogenase, which means that, in the regeneration reactionwhich reconverts the oxidized cofactor into its original reduced form,2-propanol is oxidized to acetone by means of an alcohol dehydrogenase.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that oxygen is used whichis reduced by means of an NADH oxidase.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that, as a secondaryalcohol, malate (cosubstrate) is used which is oxidized to pyruvate andCO₂ by means of an oxaloacetate-decarboxylating malate dehydrogenase(“malate enzyme”), e.g., that in the regeneration reaction whichreconverts the oxidized cofactor into its original reduced form, malateis oxidized to pyruvate and CO₂ by means of a malate dehydrogenase.

In this embodiment, the nascent pyruvate is reacted in a further redoxreaction which does not serve for the formation of a product, butconstitutes the second cofactor regeneration reaction.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that it is used forperforming at least one oxidation reaction and at least one reductionreaction, respectively, in the same reaction batch on compounds ofgeneral formula

wherein

R₄ denotes hydrogen, a methyl group, a hydroxy group or an oxo group,

R₅ denotes hydrogen, a hydroxy group, an oxo group or a methyl group,

R₆ denotes hydrogen or a hydroxy group,

R₇ denotes hydrogen, —COR₁₃, wherein R₁₃ is a C₁-C₄ alkyl group which isunsubstituted or substituted with a hydroxy group, or a C₁-C₄ carboxyalkyl group which is substituted, in particular with a hydroxy group, orunsubstituted,

or

R₆ and R₇ together denote an oxo group,

R₈ denotes hydrogen, a methyl group, a hydroxy group or an oxo group,

R₉ denotes hydrogen, a methyl group, a hydroxy group or an oxo group,

R₁₀ denotes hydrogen, a methyl group or a halogen,

R₁₁ denotes hydrogen, a methyl group, a hydroxy group, an oxo group orhalogen, and

R₁₂ denotes hydrogen, a hydroxy group, an oxo group or a methyl group,

wherein the structural element

denotes a benzene ring or a ring comprising 6 carbon atoms and 0, 1 or 2C—C-double bonds;

wherein the substrate(s) is/are preferably provided at a concentrationof <5% (w/v) in the reaction batch for the reduction reaction(s)involved in the formation of the product.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that an enzymaticconversion of dehydroepiandrosterone (DHEA) of formula

into testosterone of formula

takes place.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that an enzymaticepimerization of the hydroxysteroid compound 3α,7α-dihydroxy-5β-cholanicacid (chenodeoxycholic acid, CDC) of formula

occurs via oxidation to ketolithocholic acid (KLC) of formula

and reduction to 3α,7β-dihydroxy-5β-cholanic acid (ursodeoxycholic acid,UDC) of formula

e.g. using two opposite stereospecific hydroxysteroid dehydrogenases.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that it is used for theenzymatic epimerization of 3α,7α,12α-trihydroxy-5β-cholanic acid(cholanic acid) of formula

either

-   -   A) via oxidation to obtain 3α,7α-dihydroxy-12-oxo-5β-cholanic        acid (12-oxo-CDC) of formula

which is further reacted to obtain 3α-hydroxy-7,12-dioxo-5β-cholanicacid (12oxo-KLC) of formula

and subsequent reduction to the stereoisomeric hydroxy compound3α,7β-dihydroxy-12-oxo-5β-cholanic acid (12-keto-ursodeoxycholanic acid)of formula

or

-   -   B) via oxidation to obtain 3α,12α-dihydroxy-7-oxo-5β-cholanic        acid of formula

followed by enzymatic oxidation to obtain3α-hydroxy-7,12-dioxo-5β-cholanic acid(12oxo-KLC) of formula XI, andsubsequent reduction to obtain the stereoisomeric hydroxy compound3α,7β-dihydroxy-12-oxo-5β-cholanic acid (12-keto-ursodeoxycholanic acid)of formula XII,

or

-   -   C) via oxidation to obtain 3α,12α-dihydroxy-7-oxo-5β-cholanic        acid of formula XIII, followed by enzymatic reduction to obtain        3α,7β,12α-triydroxy-5β-cholanic acid of formula

and subsequent oxidation to obtain the stereoisomeric hydroxy compound3α,7β-dihydroxy-12-oxo-5β-cholanic acid (12-keto-ursodeoxycholanic acid)of formula XII;

or

in any combination from A), B) and/or C)

e.g. using 3 stereospecific hydroxysteroid dehydrogenases, 2 of whichhave opposite stereospecifity.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that a C₅- or C₆-sugar isused as a substrate, e.g., that the process is used for theisomerization of C₅- or C₆-sugars.

In a preferred embodiment of the present invention, a process accordingto the present invention is characterized in that an isomerization ofglucose occurs via reduction to sorbitol and oxidation to fructose,e.g., that the process is used for the isomerization of glucose viareduction to sorbitol and subsequent oxidation to fructose.

A process according to the present invention is preferably carried outin an aqueous system, wherein it is possible that the substrate for theoxidation and reduction reaction is partly provided in an undissolvedstate in the form of a suspension and/or as a second liquid phase.

In a particular embodiment, a process according to the present inventionis characterized in that the substrate(s) for the oxidation reaction(s)involved in the formation of a product is/are provided in the reactionbatch at a concentration of at least 5% (w/v) and more, preferably 7%(w/v) and more, particularly preferably 9% (w/v) and more, e.g. 5% (w/v)to 20% (w/v), such as 5% (w/v) to 15% (w/v), e.g. 5% (w/v) to 12% (w/v),such as 5% (w/v) to 10% (w/v).

According to a preferred embodiment of the process according to theinvention, the substrate(s), which substrate(s) are used as substrate(s)of the product-forming reactions, are initially present in the reactionbatch at a concentration of ≥3% (w/v).

In a particular embodiment, a process according to the present inventionis characterized in that, on the whole, a turnover of ≥70%, inparticular ≥90%, is achieved in the product-forming reactions.

In a process according to the present invention, a buffer can be addedto the aqueous system. Suitable buffers are, for example, potassiumphosphate, Tris-HCl and glycine with a pH ranging from 5 to 10,preferably from 6 to 9. Furthermore or alternatively, ions forstabilizing the enzymes, such as Mg²⁺ or other additives such asglycerol, can be added to the system. In a process according to thepresent invention, the concentration of the added cofactors NAD(P)⁺ andNAD(P)H is usually between 0.001 mM and 10 mM, preferably between 0.01mM and 1 mM.

Depending on the enzymes used, the process according to the presentinvention can be performed at a temperature ranging from 10° C. to 70°C., preferably from 20° C. to 45° C.

Hydroxysteroid dehydrogenases (HSDH) are understood to be enzymes whichcatalyze the oxidation of hydroxy groups to the corresponding ketogroups or, conversely, the reduction of keto groups to the correspondinghydroxy groups at the steroid skeleton.

Appropriate hydroxysteroid dehydrogenases which can be used for redoxreactions on hydroxysteroids are, for example, 3α-HSDH, 3β-HSDH,7α-HSDH, 7β-HSDH or 17β-HSDH.

Appropriate enzymes with 7α-HSDH activity can be obtained, for example,from Clostridia (Clostridium absonum, Clostridium sordelii), Escherichiacoli or Bacteroides fragilis.

Appropriate enzymes with 7β-HSDH activity can be obtained, for example,from Ruminococcus sp. or Clostridium absonum.

Appropriate lactate dehydrogenases can be obtained, for example, fromOryctolagus cuniculus.

Appropriate alcohol dehydrogenases can be obtained, for example, fromLactobacillus kefir.

An appropriate xylose reductase can be obtained, for example, fromCandida tropicalis.

Appropriate sorbitol dehydrogenases can be obtained, for example, fromsheep liver, Bacillus subtilis or Malus domestica.

Appropriate NADH oxidases can be obtained, for example, from Leuconostocmesenteroides, Streptococcus mutans, Clostridium aminovalericum.

In a process according to the present invention, enzymes are preferablyused as proteins recombinantly overexpressed in E. coli, wherein thecorresponding cell lysates preferably continue to be used without anyfurther purification. Thereby, the enzyme unit 1 U corresponds to theenzyme amount which is required for reacting 1 μmol of substrate permin.

EXAMPLES

In the following examples, all temperature data are given in degreesCelsius (° C.). The following abbreviations are used:

EtOAc=ethyl acetate

h=hour(s)

IPA=isopropyl alcohol (2-propanol)

MeOH=methanol

Rt=room temperature

Example 1 Epimerization of Chenodeoxycholic Acid into UrsodeoxycholicAcid by 7α-hydroxysteroid Dehydrogenase and 7β-hydroxysteroidDehydrogenase, Using a Lactate Dehydrogenase- and AlcoholDehydrogenase-Dependent Cofactor Regeneration System

A 0.5 ml charge contains 50 mg chenodeoxycholic acid, 12 U ofrecombinant 7α-hydroxysteroid dehydrogenase from Escherichia coli, 6 Uof recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcus torquesas well as 0.5 mM NAD⁺ and 0.3 mM NADPH. For the regeneration of NAD⁺, 6U of recombinant lactate dehydrogenase and 350 mM sodium pyruvate areused. For the regeneration of NADPH, 6 U of recombinant alcoholdehydrogenase from Lactobacillus kefir and initially 2.4% IPA (w/v) areused. The reaction is performed in an aqueous potassium phosphate buffer(100 mM, pH=7.8) at 25° C., with continuous shaking (850 rpm). An opensystem continues to be used in order to facilitate the evaporation ofacetone and to shift the reaction toward ursodeoxycholic acid. 1.6%(w/v) IPA is additionally dosed in after 6 h, 2.4% (w/v) IPA after 16 h,3.9% (w/v) IPA after 24 h and 0.8% (w/v) IPA after 40 h. In addition, 20μl of 4-methyl-2-pentanol is added after 24 h. 200 μl of 2-pentanol aswell as 1.6% (w/v) IPA are added after 46 h. After 48 h, the proportionof ursodeoxycholic acid in all bile acids in the reaction mixture is>97%.

Example 2 Epimerization of Chenodeoxycholic Acid into UrsodeoxycholicAcid by 7α-hydroxysteroid Dehydrogenase and 7β-hydroxysteroidDehydrogenase, Using a Lactate Dehydrogenase- and MalateDehydrogenase-Dependent Cofactor Regeneration System

A 0.5 ml charge contains 50 mg chenodeoxycholic acid, 20 U ofrecombinant 7α-hydroxysteroid dehydrogenase from Escherichia coli, 20 Uof recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcus torquesas well as 1 mM NAD⁺ and 1 mM NADPH. For the regeneration of NAD⁺, 10 Uof the lactate dehydrogenase (Sigma-Aldrich) are used, and for startingthe reaction, 16.5 mM sodium pyruvate is used. For the regeneration ofNADPH, 20 U of recombinant malate dehydrogenase from Escherichia coliand 320 mM sodium malate are used. The reaction is performed in anaqueous potassium phosphate buffer (100 mM, pH=7.8) at 25° C., withcontinuous shaking (850 rpm). An open system continues to be used inorder to allow the nascent CO₂ to escape. 20 U of 7α-HSDH as well as 10U of lactate dehydrogenase were additionally dosed in after 16 h andafter 40 h. 10 U 7β-HSDH were additionally dosed in after 20 h, 24 h, 44h and 48 h. Furthermore, 10 U of malate dehydrogenase were additionallydosed in after 40 h. After 72 h, the proportion of ursodeoxycholic acidin all bile acids in the reaction mixture is approximately 90%.

Example 3 Epimerization of Chenodeoxycholic Acid into UrsodeoxycholicAcid by 7α-hydroxysteroid Dehydrogenase and 7β-hydroxysteroidDehydrogenase, Using an NADH Oxidase- and AlcoholDehydrogenase-Dependent Cofactor Regeneration System

A 0.5 ml charge contains 50 mg chenodeoxycholic acid, 12 U ofrecombinant 7α-hydroxysteroid dehydrogenase from Escherichia coli, 7.5 Uof recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcus torquesas well as 1 mM NAD⁺ and 1 mM NADPH. For the regeneration of NAD⁺, 20 Uof recombinant NADH oxidase from Clostridium aminovalericum are used.For the regeneration of NADPH, 5 U of recombinant alcohol dehydrogenasefrom Lactobacillus kefir and initially 2% IPA (w/v) are used. Thereaction is performed in an aqueous potassium phosphate buffer (100 mM,pH 6) at 25° C., with continuous shaking (850 rpm). An open systemcontinues to be used in order to facilitate the evaporation of acetoneand to shift the reaction toward ursodeoxycholic acid. 2% IPA isadditionally dosed in after 18 h, 22 h, 26 h and 41 h as well as 5% IPAafter 41 h and after 48 h. 20 U of NADH oxidase are additionally dosedin after 24 h, and 7.5 U of 7β-hydroxysteroid dehydrogenase as well as 5U of alcohol dehydrogenase are additionally dosed in after 41 h. After48 h, the proportion of ursodeoxycholic acid in all bile acids in thereaction mixture is approximately 95-98%.

Example 4 Reprocessing and Analytics of Bile Acids

Upon completion of reactions as described in Examples 1 to 3, thereaction mixture is extracted with EtOAc. Subsequently, the solvent isremoved by evaporation. The evaporation residue is dissolved in amixture of MeOH:acetonitrile:sodium phosphate buffer pH=3, 0.78 g/l(40:30:37) and the conversion of chenodeoxycholic acid intoursodeoxycholic acid is monitored by HPLC. Thereby, a reversed-phaseseparation column (ZORBAX®Eclipse® XDB C18, flow 0.8 ml/min) and alight-refraction detector (RID), Agilent 1260 Infinity®, both fromAgilent Technologies Inc., are used.

Example 5 Conversion of Glucose into Fructose Via a Xylose Reductase anda Sorbitol Dehydrogenase, Using an Alcohol Dehydrogenase for Recyclingthe NADPH and a Lactate Dehydrogenase for Recycling the NAD⁺

A 0.5 ml charge contains 50 mg/ml glucose and 6 U/ml of recombinantxylose reductase from Candida tropicalis (overexpressed in E. coli BL21(DE3)) and 0.1 mM NADP⁺. For the regeneration of the cofactor, 7% IPAand the recombinant alcohol dehydrogenase from Lactobacillus kefir(overexpressed in E. coli BL21 (DE3)) are added. The enzymes are used inthe form of cell lysates. The reaction takes place for 24 h at 40° C.and pH=9 (50 mM Tris HCl-buffer) in an open system, with continuousshaking (900 rpm). The open system leads to the removal of acetone,which drives the reaction toward the formation of sorbitol. In the opensystem, water and IPA evaporate too, so that they are additionally dosedin after 6 h and after 21 h. Thereby at each time a total volume of 0.5ml as well as an IPA concentration of 7% (v/v) is adjusted. After 24 h,the reaction vessel is incubated at 60° C. under vacuum in order toinactivate the enzymes and to evaporate the organic solvents. Aftercooling to room temperature, the recombinant sorbitol dehydrogenase fromBacillus subtilis (overexpressed in E. coli BL21 (DE3)) is added at afinal concentration of 5 U/ml, ZnCl₂ at a final concentration of 1 mMand NAD⁺ at a final concentration of 0.1 mM. For cofactor regeneration,5 U/ml (final concentration) of lactate dehydrogenase from rabbitmuscles (Sigma Aldrich) and 300 mM pyruvate are used. The charge istopped up to 0.5 ml with water. The reaction takes place for further 24h at 40° C. in a closed system with continuous shaking (900 rpm). Aconversion of D-glucose zu D-fructose of >90% is achieved.

Example 6 Conversion of Glucose into Fructose Via a Xylose Reductase anda Sorbitol Dehydrogenase, Using an Alcohol Dehydrogenase for Recyclingthe NADPH and a NADH Oxidase for Recycling the NAD⁺

A 0.5 ml charge contains 50 mg/ml glucose, 6 U/ml of recombinant xylosereductase from Candida tropicalis (overexpressed in E. coli BL21 (DE3))and 0.1 mM NADP⁺. For the regeneration of the cofactor, 7% (v/v) IPA andthe recombinant alcohol dehydrogenase from Lactobacillus kefir(overexpressed in E. coli BL21 (DE3)) are added. The enzymes are used inthe form of cell lysates. The reaction takes place for 24 h at 40° C.and pH=8 (50 mM Tris HCl buffer) in an open system, with continuousshaking (900 rpm). The open system leads to the removal of acetone,which drives the reaction toward the formation of sorbitol. In the opensystem, water and IPA evaporate too, so that they are additionally dosedin after 6 h and after 21 h. Thereby at each time a total volume of 0.5ml as well as an IPA-concentration of 7% (v/v) are adjusted. After 24 h,the reaction vessel is incubated at 60° C. under vacuum in order toinactivate the enzymes and to evaporate IPA as well as any acetone thathas formed. After cooling to room temperature, the recombinantD-sorbitol dehydrogenase from Bacillus subtilis (overexpressed in E.coli BL21 (DE3)) is added at a final concentration of 5 U/ml, CaCl₂ at afinal concentration of 1 mM and a mixture of NAD⁺ and NADH at a finalconcentration of 0.1 mM. For cofactor regeneration, 10 U/ml (finalconcentration) of NADH oxidase from Leuconostoc mesenteroides(overexpressed in E. coli BL21 (DE3)) are used. The enzymes are used inthe form of cell lysates. The charge is topped up to 0.5 ml with water.The reaction takes place for 24 h at 40° C. in an open system, withcontinuous shaking (900 rpm), in order to ensure sufficient oxygensupply for the NADH oxidase from the air. In that open system at 40° C.water evaporates. Thus, after 6 h and after 21 h it is filled up to withwater to a volume of 0.5 ml. A conversion of D-glucose into D-fructoseof ca. 98% is achieved.

Example 7 Reprocessing and Analytics of Sugars

The charge is incubated at 65° C. for 10 min for inactivating theenzymes and is subsequently centrifuged. The supernatant is thenfiltered over a 0.2 μM PVDF filter and analyzed by ligand-exchange HPLC(Agilent Technologies Inc.). In doing so, sugars and polyols areseparated via a lead column of Showa Denko K.K. (Shodex® Sugar SP0810)with a flow of 0.5 ml/min water (VWR International GmbH, HPLC Grade) at80° C. Detection occurs with the aid of a light-refraction detector(RID, Agilent 1260 Infinity®, Agilent Technologies Inc.). An inlinefilter of Agilent Technologies Inc. and, as precolumns, ananion-exchange column (Shodex® Axpak-WAG), a reversed-phase column(Shodex® Asahipak® ODP-50 6E) and a sugar precolumn (SUGAR SP-G) ofShowa Denko K.K. are used.

Example 8 Bioconversion of Cholanic Acid to3α,7-dihydroxy-12-oxo-5-cholanic Acid by12α-hydroxysteroiddehydrogenase, 7α-hydroxysteroiddehydrogenase and7β-hydroxysteroiddehydrogenase Using a Lactate Dehydrogenase and anAlcohol Dehydrogenase Dependent Cofactor Regeneration System

A 0.5 ml charge contains 25 mg of cholanic acid 12.5 U of recombinant12α-hydroxysteroid dehydrogenase from Eggertella lenta or Lysinibacillussphaericus, 16 U of recombinant 7α-hydroxysteroid dehydrogenase fromEscherichia coli, 6 U of recombinant 7β-hydroxysteroid dehydrogenasefrom Ruminococcus torques, as well as 1 mM NAD⁺ and 1 mM NADPH. Forregeneration of NAD⁺ 12.5 U of recombinant lactate dehydrogenase fromOryctolagus cuniculus (muscle isoform) and 200 mM of sodium pyruvate areused. For regeneration of NADPH 5 U of recombinant alcohol dehydrogenasefrom Lactobacillus kefir and initially 2% of IPA (w/v) are used. Thereaction is carried out in an aqueous potassium phosphate buffer (100mM, pH 7.8) at 25° C. under continuous shaking (850 rpm). An open systemis further used in order to allow evaporation of acetone and to shiftthe reaction towards 3α,7β-dihydroxy-12-oxo-5β-cholanic acid. After 18 hand 24 h 2% IPA (w/v) are dosed in additionally. After 48 h 61% of thecholanic acid used are reacted to 3α,7α-dihydroxy-12-oxo-5β-cholanicacid.

Example 9 Bioconversion of Cholanic Acid to3α,7-dihydroxy-12-oxo-5-cholanic Acid by 12α-hydroxysteroidDehydrogenase, 7α-hydroxysteroid Dehydrogenase and 7β-hydroxysteroidDehydrogenase Using a Lactate Dehydrogenase, NADH-Oxidase and AlcoholDehydrogenase Dependent Cofactor Regeneration System

A 0.5 ml charge contains 25 mg of cholanic acid, 12.5 U of recombinant12α-hydroxysteroid dehydrogenase from Eggertella lenta or Lysinibacillussphaericus, 16 U of recombinant 7α-hydroxysteroid dehydrogenase fromEscherichia coli, 6 U of recombinant 7β-hydroxysteroid dehydrogenasefrom Ruminococcus torques, as well as 1 mM NAD⁺ and 1 mM NADPH. For theregeneration of NAD⁺ 5 U of recombinant NADH oxidase from Leuconostocmesenteroides and 12.5 U of recombinant lactate dehydrogenase fromOryctolagus cuniculus (muscle isoform) and 200 mM of sodium pyruvate areused. For the regeneration of NADPH 5 U of recombinant alcoholdehydrogenase from Lactobacillus kefir and initially 2% of IPA (w/v) areused. The reaction is carried out in an aqueous potassium phosphatebuffer (100 mM, pH 7.8) at 25° C. under continuous shaking (850 rpm).

An open system is further used in order to allow evaporation of acetoneand to shift the reaction towards 3α,7β-dihydroxy-12-oxo-5β-cholanicacid. After 18 h and 24 h 2% of IPA (w/v) are dosed in additionally.After 48 h 70% of the cholanic acid used are reacted to3α,7α-dihydroxy-12-oxo-5β-cholanic acid.

Example 10 Epimerization of Chenodeoxy Cholanic Acid into UrsodeoxyCholanic Acid Using 7α-hydroxysteroid Dehydrogenase and7β-hydroxysteroid Dehydrogenase Under Use of a Lactate Dehydrogenase andAlcohol Dehydrogenase Dependent Cofactor Regeneration System. Advantageof Adding Manganese Chlorid (MnCl₂)

A 0.5 ml charge contains 50 mg of chenodeoxy cholanic acid, 12 U ofrecombinant 7α-hydroxysteroiddehydrogenase aus Escherichia coli, 6 U ofthe recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcustorques, as well as 0.5 mM NAD⁺ and 0.3 mM NADPH. For the regenerationof NAD⁺ 6 U of recombinant lactate dehydrogenase and 350 mM of sodiumpyruvate are used. For the regeneration of NADPH 6 U of recombinantalcohol dehydrogenase from Lactobacillus kefir and initially 2.4% of IPA(w/v) are used. The reaction is carried out in an aqueous potassiumphosphate buffer (100 mM, pH=7.8) with 5 mM MnCl₂ at 25° C. and undercontinuous shaking (850 rpm). An open system is further used in order toallow evaporation of acetone and to shift the reaction towards ursodeoxycholanic acid. 1.6% (w/v) of IPA after 6 h, 2.4% (w/v) of IPA after 16 hand 3.9% (w/v) of IPA after 24 h are dosed in additionally. After 36 h200 μl of 2-pentanol as well as 3% (w/v) of IPA are added and after 48 h100 μl 2-pentanol and 4% (w/v) of IPA are dosed in additionally. After64 h the part of ursodeoxy cholanic acid of all bile acids in thereaction mixture is >99%. In particular, the part of chenodeoxy cholanicacid ca. 0.3%. In a control charge without the addition of MnCl₂ thepart of chenodeoxy cholanic acid is at ca. 2% and the part of ursodeoxycholanic acid at ca. 97.5% (average value from 5 experiments each).

Example 11

Epimerization of chenodeoxy cholanic acid to ursodeoxy cholanic acid by7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenaseunder use of an alcohol dehydrogenase dependent cofactor regenerationsystem as well as a combined lactate dehydrogenase and NADH oxidasedependent cofactor regeneration system

A 0.5 ml charge contains 50 mg of chenodeoxy cholanic acid, 12 U ofrecombinant 7α-hydroxysteroid dehydrogenase from Escherichia coli, 6 Uof recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcustorques, as well as 0.5 mM of NAD⁺ and 0.3 mM of NADPH. For theregeneration of NAD⁺ 6 U of recombinant lactate dehydrogenase and 350 mMof sodium pyruvate are used. For the regeneration of NAD⁺ in addition 9U of recombinant NADH oxidase from Leuconostoc mesenteroides, as well as6 U of recombinant NADH oxidase from Clostridium aminovalericum areused. For the regeneration of NADPH 6 U of recombinant alcoholdehydrogenase from Lactobacillus kefir and initially 2.4% (w/v) of IPAare used. The reaction is carried out in an aqueous potassium phosphatebuffer (100 mM, pH=7.8) at 25° C. under continuous shaking (850 rpm). Anopen system is further used in order to allow evaporation of acetone andto shift the reaction towards ursodeoxy cholanic acid. After 6 h 1.6%(w/v) of IPA, after 16 h 2.4% (w/v) of IPA and after 24 h 3.9% (w/v) ofIPA are dosed in additionally. After 36 h 200 μl of 2-pentanol as wellas 3% (w/v) of IPA are added and after 48 h 100 μl of 2-pentanol and 4%(w/v) of IPA are additionally dosed in. After 64 h the part of ursodeoxycholanic acid of all bile acids in the reaction mixture is >99%. Inparticular the part of chenodeoxy cholanic acid is ca. 0.2%. In acontrol charge without addition of NADH-oxidase the part of chenodeoxycholanic acid is at ca. 2% and the part of ursodeoxy cholanic acid atca. 97.5% (same control charge as in example 11; average values from 5experiments each).

Example 12 Epimerization of Chenodeoxy Cholanic Acid to UrsodeoxyCholanic Acid by 7α-hydroxysteroid Dehydrogenase and 7β-hydroxysteroidDehydrogenase Under Use of an Alcohol Dehydrogenase Dependent CofactorRegeneration System as well as a Combined Lactate Dehydrogenase and NADHOxidase Dependent Cofactor Regeneration System. Additive Effect of2-pentanol and 2-propanol

A 50 ml charge contains 5 g of chenodeoxy cholanic acid, 24 U/ml ofrecombinant 7α-hydroxysteroid dehydrogenase from Escherichia coli, 12U/ml of recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcustorques as well as 055 mM of NAD⁺ and 0.3 mM of NADPH. For theregeneration of NAD⁺ 12 U/ml recombinant lactate dehydrogenase and 350mM of sodium pyruvate are used. For the regeneration of NAD⁺additionally 18 U/ml of recombinant NADH oxidase from Leuconostocmesenteroides as well as 12 U/ml of recombinant NADH oxidase fromClostridium aminovalericum are used. For the regeneration of NADPH 12U/ml of recombinant Alcohol dehydrogenase from Lactobacillus kefir andinitially 1.5% (w/v) of IPA are used. The reaction is carried out in anaqueous potassium phosphate buffer (100 mM, pH=7.8) with 5 mM MnCl₂ at25° C. In a 3-neck-piston it is stirred with a KPG-stirrer at ca. 100rpm. Removal of acetone which originates from the reaction is effectedby a stream of air (ca. 400-600 ml/min) through the reaction vessel.Since at the same time 2-propanol is evaporated as well, additionaldosing is necessary, e.g. in an amount of 0.75 ml (1.5 h), 0.75 ml (3h), 0.5 ml (4 h), 0.75 ml (6 h), 0.75 ml (8 h), 0.5 ml (11 h), 0.5 ml(14 h), 0.5 ml (17 h), 0.5 ml (21 h), 1 ml (23 h), 2.5 ml (25 h), 4 ml(29 h). After ca. 30 h, 20 ml 2-pentanol as well as 2 ml 2-propanol wereadded. After 46 h of total reaction time the part of 7-ketolithocholanicacid is ca. 1% (related to the sum of chenodeoxy cholanic acid,ursodeoxy cholanic acid and 7-ketolithocholanic acid. Further 2-propanolis added: 3 ml (46 h), 4 ml (52 h), 4 ml (54 h), as well as in addition10 ml of 2-pentanol. After 72 h reaction time in total the part of7-ketolithocholanic acid can be lowered to less than 0.2%. The part ofursodeoxy cholanic acid is >99%.

Example 13 Workup and Analytics of Bile Acids

After termination of the reaction as described in examples 8 to 12, thebile acids which are present in the trials may be analyzed via a methodas described in example 4.

The invention claimed is:
 1. A process for the enzymatic regeneration ofthe redox cofactors NAD⁺/NADH and NADP⁺/NADPH in a one-pot reaction,wherein, as a result of at least two further enzymatically catalysedredox reactions proceeding in the same reaction batch (product-formingreactions), the redox cofactor ⁺/NADH accumulates in its reduced form asNADH and the redox cofactor NADP⁺/NADPH accumulates in its oxidized formas NADP⁺, wherein: a) in the regeneration reaction which reconverts NADHinto its original oxidized form, oxygen or pyruvate is reduced by meansof an NADH oxidase or a lactate dehydrogenase, and b) in theregeneration reaction which reconverts NADP⁺ into its original reducedform, 2-propanol or malate is oxidized by means of an alcoholdehydrogenase or a malate dehydrogenase.
 2. The process according toclaim 1, wherein oxidation reaction(s) and reduction reaction(s) takeplace on the same substrate (molecular backbone).
 3. The processaccording to claim 1, wherein oxidation reaction(s) and reductionreaction(s) proceed chronologically parallel.
 4. The process accordingto claim 1, wherein, in the regeneration reaction which reconverts NADP+to NADPH, 2-propanol is oxidized to acetone by means of an alcoholdehydrogenase.
 5. The process according to claim 1, wherein, in theregeneration reaction which reconverts NADH to NAD+, pyruvate is reducedto lactate by means of a lactate dehydrogenase.
 6. The process accordingto claim 5, wherein, in the regeneration reaction which reconverts NADP+to NADPH, malate is oxidized to pyruvate and CO₂ by means of a malatedehydrogenase.
 7. The process according to claim 1, wherein the processis used for performing at least one oxidation reaction and at least onereduction reaction, respectively, in the same reaction batch oncompounds of general formula

wherein R₄ denotes hydrogen, a methyl group, a hydroxy group or an oxogroup, R₅ denotes hydrogen, a hydroxy group, an oxo group or a methylgroup, R₆ denotes hydrogen or a hydroxy group, R₇ denotes hydrogen,CORD, wherein R₁₃ is a C₁-C₄ alkyl group which is unsubstituted orsubstituted with a hydroxy group, or a C₁-C₄ carboxy alkyl group whichis substituted or unsubstituted, or R₆ and R₇ together denote an oxogroup, R₈ denotes hydrogen, a methyl group, a hydroxy group or an oxogroup, R₉ denotes hydrogen, a methyl group, a hydroxy group or an oxogroup, R₁₀ denotes hydrogen, a methyl group or halogen, R₁₁ denoteshydrogen, a methyl group, a hydroxy group, an oxo group or halogen, andR₁₂ denotes hydrogen, a hydroxy group, an oxo group or a methyl group,wherein the structural element

denotes a benzene ring or a ring comprising 6 carbon atoms and 0, 1 or 2C—C-double bonds.
 8. The process according to claim 7, wherein R₇denotes —COR₁₃, wherein R₁₃ is a C₁-C₄ carboxy alkyl group which issubstituted with a hydroxy group.
 9. The process according to claim 7,wherein it is used for the conversion of dehydroepiandrosterone offormula

into testosterone of formula


10. The process according to claim 7, wherein it is used for theenzymatic epimerization of 3α,7α-dihydroxy-5β-cholanic acid of formula

into ketolithocholic acid of formula

by oxidation, and into the stereoisomeric hydroxy compound3α,7β-dihydroxy-5β-cholanic acid of formula

by subsequent reduction, using two opposite stereospecifichydroxysteroid dehydrogenases.
 11. The process according to claim 10,whereby the oxidation reaction is catalysed by a 7α-hydroxysteroiddehydrogenase from E. coli; and/or the reduction reaction is catalysedby a 7β-hydroxysteroid dehydrogenase from Ruminococcus torques.
 12. Theprocess according to claim 7, wherein it is used for the enzymaticepimerization of 3α,7α,12α-trihydroxy-5β-cholanic acid (cholanic acid)of formula

either A) via oxidation to obtain 3α,7α-dihydroxy-12-oxo-5β-cholanicacid (12-oxo-CDC) of formula

which is further reacted to obtain 3α-hydroxy-7,12-dioxo-5β-cholanicacid (12oxo-KLC) of formula

and subsequent reduction to the stereoisomeric hydroxy compound3α,7β-dihydroxy-12-oxo-5β-cholanic acid (12-keto-ursodeoxycholanic acid)of formula

or B) via oxidation to obtain 3α,12α-dihydroxy-7-oxo-5β-cholanic acid offormula

followed by enzymatic oxidation to obtain3α-hydroxy-7,12-dioxo-5β-cholanic acid (12oxo-KLC) of formula XI, andsubsequent reduction to obtain the stereoisomeric hydroxy compound3α,7β-dihydroxy-12-oxo-5β-cholanic acid (12-keto-ursodeoxycholanic acid)of formula XII, or C) via oxidation to obtain3α,12α-dihydroxy-7-oxo-5β-cholanic acid of formula XIII, followed byenzymatic reduction to obtain 3α,7β,12α-trihydroxy-5β-cholanic acid offormula

and subsequent oxidation to obtain the stereoisomeric hydroxy compound3α,7β-dihydroxy-12-oxo-5β-cholanic acid (12-keto-ursodeoxycholanic acid)of formula XII; using 3 stereospecific hydroxysteroid dehydrogenases, 2of which have opposite stereospecificity.
 13. The process according toclaim 1, wherein it is used for the isomerization of C₅- or C₆-sugars.14. The process according to claim 13 for the isomerization of glucosevia reduction to sorbitol and subsequent oxidation to fructose.
 15. Theprocess according to claim 1, wherein the substrate(s) for the oxidationreaction(s) involved in the formation of a product is/are provided inthe reaction batch at a concentration of at least 5% (w/v) and more. 16.The process according to claim 1, wherein, on the whole, a turnover of≥70% is achieved in the two further enzymatically catalysed redoxreactions proceeding in the same reaction batch.
 17. The processaccording to claim 16, wherein, on the whole, a turnover of ≥90% isachieved in the two further enzymatically catalysed redox reactionsproceeding in the same reaction batch.